Methods of reducing apolipoprotein C-III

ABSTRACT

In various embodiments, the present invention provides methods of treating and/or preventing cardiovascular-related disease and, in particular, a method of blood lipid therapy comprising administering to a subject in need thereof a pharmaceutical composition comprising eicosapentaenoic acid or a derivative thereof.

PRIORITY CLAIM

This application claims priority to U.S. provisional patent applicationNo. 61/761,399, filed on Feb. 6, 2013, and U.S. provisional patentapplication No. 61/809,138, filed on Apr. 5, 2013, the contents of eachof which are incorporated by reference herein and relied upon.

BACKGROUND

Cardiovascular disease is one of the leading causes of death in theUnited States and most European countries. It is estimated that over 70million people in the United States alone suffer from a cardiovasculardisease or disorder including but not limited to high blood pressure,coronary heart disease, dyslipidemia, congestive heart failure andstroke. A need exists for improved treatments for cardiovasculardiseases and disorders.

SUMMARY

In various embodiments, the present invention provides methods oftreating and/or preventing cardiovascular-related diseases and, inparticular, a method of blood lipid therapy comprising administering toa subject in need thereof a pharmaceutical composition comprisingeicosapentaenoic acid or a derivative thereof. In one embodiment, themethod comprises administering to a subject about 2 g to about 4 g perday (e.g., about 3.8 g to about 4 g per day) of eicosapentaenoic acid orderivative thereof.

In one embodiment, the composition contains not more than 10%, byweight, docosahexaenoic acid or derivative thereof, substantially nodocosahexaenoic acid or derivative thereof, or no docosahexaenoic acidor derivative thereof. In another embodiment, eicosapentaenoic acidethyl ester comprises at least 90%, by weight, of all fatty acidspresent in the composition. In one embodiment, the present inventionprovides methods of reducing the level of apolipoprotein C-III (alsoreferred to as Apolipoprotein C3 or ApoC3) in a subject compared tobaseline or to placebo control.

In one embodiment, a pharmaceutical composition useful in accordancewith the invention comprises, consists of or consists essentially of atleast 95% by weight ethyl eicosapentaenoate (EPA-E), about 0.2% to about0.5% by weight ethyl octadecatetraenoate (ODTA-E), about 0.05% to about0.25% by weight ethyl nonaecapentaenoate (NDPA-E), about 0.2% to about0.45% by weight ethyl arachidonate (AA-E), about 0.3% to about 0.5% byweight ethyl eicosatetraenoate (ETA-E), and about 0.05% to about 0.32%ethyl heneicosapentaenoate (HPA-E). In another embodiment, thecomposition is present in a capsule shell. In another embodiment, thecomposition contains substantially no or no amount of docosahexaenoicacid (DHA) or derivative thereof such as ethyl-DHA (DHA-E).

In another embodiment, the invention provides a method of treatingmoderate to severe hypertriglyceridemia comprising administering acomposition as described herein to a subject in need thereof one toabout four times per day.

These and other embodiments of the present invention will be disclosedin further detail herein below.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1 depicts reductions in total apolipoprotein C3 values for subjectshaving fasting triglyceride levels≧500 mg/dL (solid) and subjects onstatin therapy having fasting triglyceride levels of 200-499 mg/dL(hashed) after being administered 2 g/day or 4 g/day of a compositionaccording to the present invention (AMR101).

DETAILED DESCRIPTION

While the present invention is capable of being embodied in variousforms, the description below of several embodiments is made with theunderstanding that the present disclosure is to be considered as anexemplification of the invention, and is not intended to limit theinvention to the specific embodiments illustrated. Headings are providedfor convenience only and are not to be construed to limit the inventionin any manner. Embodiments illustrated under any heading may be combinedwith embodiments illustrated under any other heading.

The use of numerical values in the various quantitative values specifiedin this application, unless expressly indicated otherwise, are stated asapproximations as though the minimum and maximum values within thestated ranges were both preceded by the word “about.” Also, thedisclosure of ranges is intended as a continuous range including everyvalue between the minimum and maximum values recited as well as anyranges that can be formed by such values. Also disclosed herein are anyand all ratios (and ranges of any such ratios) that can be formed bydividing a disclosed numeric value into any other disclosed numericvalue. Accordingly, the skilled person will appreciate that many suchratios, ranges, and ranges of ratios can be unambiguously derived fromthe numerical values presented herein and in all instances such ratios,ranges, and ranges of ratios represent various embodiments of thepresent invention.

In one embodiment, the invention provides a method for treatment and/orprevention of a cardiovascular-related disease. The term“cardiovascular-related disease” herein refers to any disease ordisorder of the heart or blood vessels (i.e. arteries and veins) or anysymptom thereof. Non-limiting examples of cardiovascular-related diseaseand disorders include hypertriglyceridemia, hypercholesterolemia, mixeddyslipidemia, coronary heart disease, vascular disease, stroke,atherosclerosis, arrhythmia, hypertension, myocardial infarction, andother cardiovascular events.

The term “treatment” in relation a given disease or disorder, includes,but is not limited to, inhibiting the disease or disorder, for example,arresting the development of the disease or disorder; relieving thedisease or disorder, for example, causing regression of the disease ordisorder; or relieving a condition caused by or resulting from thedisease or disorder, for example, relieving, preventing or treatingsymptoms of the disease or disorder. The term “prevention” in relationto a given disease or disorder means: preventing the onset of diseasedevelopment if none had occurred, preventing the disease or disorderfrom occurring in a subject that may be predisposed to the disorder ordisease but has not yet been diagnosed as having the disorder ordisease, and/or preventing further disease/disorder development ifalready present.

In one embodiment, the present invention provides a method of bloodlipid therapy comprising administering to a subject or subject group inneed thereof a pharmaceutical composition as described herein. Inanother embodiment, the subject or subject group hashypertriglyceridemia, hypercholesterolemia, mixed dyslipidemia and/orvery high triglycerides.

In another embodiment, the subject or subject group being treated has abaseline triglyceride level (or mean or median baseline triglyceridelevel in the case of a subject group), fed or fasting, of about 200mg/dl to about 499 mg/dl. In another embodiment, the subject or subjectgroup has a baseline LDL-C level (or mean or median baseline LDL-Clevel), despite stable statin therapy, of about 40 mg/dl to about 115 orabout 40 to about 100 mg/dl.

In one embodiment, the subject or subject group being treated inaccordance with methods of the invention is on concomitant statintherapy, for example atorvastatin, rosuvastatin or simvastatin therapy(with or without ezetimibe). In another embodiment, the subject is onconcomitant stable statin therapy at time of initiation of EPA therapy.

In another embodiment, the subject or subject group being treated inaccordance with methods of the invention has a body mass index (BMI ormean BMI) of not more than about 45 kg/m².

In one embodiment, the invention provides a method of loweringtriglycerides in a subject on stable statin therapy having baselinefasting triglycerides of about 200 mg/dl to about 499 mg/dl, the methodcomprising administering to the subject a pharmaceutical compositioncomprising about 1 g to about 4 g of EPA or a derivative thereof,wherein upon administering the composition to the subject daily for aperiod of 1 to about 12 weeks the subject exhibits at least 10%, atleast 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, or at least 75% lower fasting triglyceridesthan a control subject maintained on stable statin therapy (andoptionally placebo matching the EPA) without concomitant EPA for aperiod of 1 to about 12 weeks, wherein the control subject also hasbaseline fasting triglycerides of about 200 mg/dl to about 500 mg/dl.The term “stable statin therapy” herein means that the subject, subjectgroup, control subject or control subject group in question has beentaking a stable daily dose of a statin (e.g. atorvastatin, rosuvastatinor simvastatin) for at least 4 weeks prior to the baseline fastingtriglyceride measurement (the “qualifying period”). For example, asubject or control subject on stable statin therapy would receive aconstant daily (i.e. the same dose each day) statin dose for at least 4weeks immediately prior to baseline fasting triglyceride measurement. Inone embodiment, the subject's and control subject's LDL-C is maintainedbetween about 40 mg/dl and about 115 mg/dl or about 40 mg/dl to about100 mg/dl during the qualifying period. The subject and control subjectare then continued on their stable statin dose for the 12 week periodpost baseline.

In one embodiment, the statin is administered to the subject and thecontrol subject in an amount of about 1 mg to about 500 mg, about 5 mgto about 200 mg, or about 10 mg to about 100 mg, for example about 1 mg,about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg,about 8 mg, about 9 mg, or about 10 mg; about 15 mg, about 20 mg, about25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg,about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about80 mg, about 90 mg, about 100 mg, about 125 mg, about 150 mg, about 175mg, about 200 mg, about 225 mg, about 250 mg, about 275 mg, about 300mg, about 325 mg, about 350 mg, about 375 mg, about 400 mg, about 425mg, about 450 mg, about 475 mg, or about 500 mg. In another embodiment,the subject (and optionally the control subject) has a baseline LDL-Clevel, despite stable statin therapy, of about 40 mg/dl to about 115mg/dl or about 40 mg/dl to about 100 mg/dl. In another embodiment, thesubject and/or control subject has a body mass index (BMI; or mean BMI)of not more than about 45 kg/m².

In another embodiment, the invention provides a method of loweringtriglycerides in a subject group on stable statin therapy having meanbaseline fasting triglycerides of about 200 mg/dl to about 499 mg/dl,the method comprising administering to members of the subject group apharmaceutical composition comprising about 1 g to about 4 g of EPA perday, wherein upon administering the composition to the members of thesubject group daily for a period of 1 to about 12 weeks the subjectgroup exhibits at least 10%, at least 15%, at least 20%, at least 25%,at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75% lowermean fasting triglycerides than a control subject group maintained onstable statin therapy without concomitant EPA (optionally with matchingplacebo) for a period of 1 to about 12 weeks, wherein the controlsubject group also has mean baseline fasting triglycerides of about 200mg/dl to about 499 mg/dl. In a related embodiment, the stable statintherapy will be sufficient such that the subject group has a mean LDL-Clevel about at least about 40 mg/dl and not more than about 100 mg/dl orabout 40 mg/dl to about 100 mg/dl for the 4 weeks immediately prior tothe baseline fasting triglyceride measurement.

In another embodiment, the invention provides a method of loweringtriglycerides in subject group on stable statin therapy and having amean baseline fasting triglyceride level of about 200 mg/dl to about 499mg/dl, the method comprising administering to members of the subjectgroup a pharmaceutical composition comprising about 1 g to about 4 g ofEPA, wherein upon administering the composition to members of thesubject group daily for a period of 1 to about 12 weeks the subjectgroup exhibits: (a) at least 10%, at least 15%, at least 20%, at least25%, at least 30%, at least 35%, at least 40%, at least 45%, at least50%, at least 55%, at least 60%, at least 65%, at least 70%, at least75% lower mean fasting triglycerides by comparison with a controlsubject group maintained on stable statin therapy without concomitantEPA (optionally with matching placebo) for a period of 1 to about 12weeks, and (b) no serum LDL-C increase, no statistically significantserum LDL-C increase, a serum LDL-C decrease, or the subject isstatistically non-inferior to the control subjects (statin plus optionalplacebo) in regard to serum LDL-C elevation) no increase in mean serumLDL-C levels compared to baseline, wherein the control subject also hasmean baseline fasting triglycerides of about 200 mg/dl to about 499mg/dl.

In another embodiment, the invention provides a method of loweringtriglycerides in subject on stable statin therapy and having meanbaseline fasting triglyceride level of about 200 mg/dl to about 499mg/dl, the method comprising administering to the subject apharmaceutical composition comprising about 1 g to about 4 g of EPA,wherein upon administering the composition to the subject daily for aperiod of 1 to about 12 weeks the subject exhibits (a) at least 10%, atleast 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, or at least 75% lower fasting triglycerides bycomparison with a control subject maintained on stable statin therapywithout concomitant EPA for a period of 1 to about 12 weeks and (b) noincrease in serum LDL-C levels compared to baseline, wherein the controlsubject also has baseline fasting triglycerides of about 200 mg/dl toabout 499 mg/dl.

In another embodiment, the invention provides a method of loweringtriglycerides in subject group on stable statin therapy and having meanbaseline fasting triglyceride level of about 200 mg/dl to about 499mg/dl, the method comprising administering to members of the subjectgroup a pharmaceutical composition comprising about 1 g to about 4 g ofEPA, wherein upon administering the composition to the members of thesubject group daily for a period of 1 to about 12 weeks the subjectgroup exhibits: (a) at least 10%, at least 15%, at least 20%, at least25%, at least 30%, at least 35%, at least 40%, at least 45%, at least50%, at least 55%, at least 60%, at least 65%, at least 70%, at least75% lower mean fasting triglycerides and (b) at least 5%, at least 10%,at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45% or at least 50% lower mean serum LDL-C levels bycomparison with a control subject group maintained on stable statintherapy without concomitant EPA (optionally with matching placebo) for aperiod of 1 to about 12 weeks, no serum LDL-C increase, no statisticallysignificant serum LDL-C increase, no statistically significant serumLDL-C increase, a serum LDL-C decrease, or the subject group isstatistically non-inferior to the control subject group (statin plusoptional placebo) in regard to serum LDL-C elevation), wherein thecontrol subject group also has mean baseline fasting triglycerides ofabout 200 mg/dl to about 499 mg/dl.

In another embodiment, the invention provides a method of loweringtriglycerides in subject group on stable statin therapy and having meanbaseline fasting triglyceride level of about 200 mg/dl to about 499mg/dl, the method comprising administering to members of the subjectgroup a pharmaceutical composition comprising about 1 g to about 4 g ofEPA, wherein upon administering the composition to the members of thesubject group daily for a period of 1 to about 12 weeks the subjectgroup exhibits (a) at least 10%, at least 15%, at least 20%, at least25%, at least 30%, at least 35%, at least 40%, at least 45%, at least50%, at least 55%, at least 60%, at least 65%, at least 70%, at least75% lower mean fasting triglycerides and (b) at least 5%, at least 10%,at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45% or at least 50% lower mean serum LDL-C levels bycomparison with a control subject group maintained on stable statintherapy without concomitant EPA (optionally with matching placebo) for aperiod of 1 to about 12 weeks, no serum LDL-C increase, no statisticallysignificant serum LDL-C increase, no statistically significant serumLDL-C increase, a serum LDL-C decrease, or the subject group isstatistically non-inferior to the control subject group (statin plusoptional placebo) in regard to serum LDL-C elevation), wherein thecontrol subject group also has mean baseline fasting triglycerides ofabout 200 mg/dl to about 500 mg/dl.

In another embodiment, the subject or subject group being treated inaccordance with methods of the invention exhibits a fasting baselineabsolute plasma level of free total fatty acid (or mean thereof) notgreater than about 300 nmol/ml, not greater than about 250 nmol/ml, notgreater than about 200 nmol/ml, not greater than about 150 nmol/ml, notgreater than about 100 nmol/ml, or not greater than about 50 nmol/ml.

In another embodiment, the subject or subject group being treated has abaseline triglyceride level (or median baseline triglyceride level inthe case of a subject group), fed or fasting, of at least about 300mg/dl, at least about 400 mg/dl, at least about 500 mg/dl, at leastabout 600 mg/dl, at least about 700 mg/dl, at least about 800 mg/dl, atleast about 900 mg/dl, at least about 1000 mg/dl, at least about 1100mg/dl, at least about 1200 mg/dl, at least about 1300 mg/dl, at leastabout 1400 mg/dl, or at least about 1500 mg/dl, for example about 200mg/dl to about 499 mg/dl, about 400 mg/dl to about 2500 mg/dl, about 450mg/dl to about 2000 mg/dl or about 500 mg/dl to about 1500 mg/dl.

In one embodiment, the subject or subject group being treated inaccordance with methods of the invention has previously been treatedwith Lovaza® and has experienced an increase in, or no decrease in,LDL-C levels and/or non-HDL-C levels. In one such embodiment, Lovaza®therapy is discontinued and replaced by a method of the presentinvention.

In another embodiment, the subject or subject group being treated inaccordance with methods of the invention exhibits a fasting baselineabsolute plasma level of free EPA (or mean thereof in the case of asubject group) not greater than about 0.70 nmol/ml, not greater thanabout 0.65 nmol/ml, not greater than about 0.60 nmol/ml, not greaterthan about 0.55 nmol/ml, not greater than about 0.50 nmol/ml, notgreater than about 0.45 nmol/ml, or not greater than about 0.40 nmol/ml.In another embodiment, the subject or subject group being treated inaccordance with methods of the invention exhibits a baseline fastingplasma level (or mean thereof) of free EPA, expressed as a percentage oftotal free fatty acid, of not more than about 3%, not more than about2.5%, not more than about 2%, not more than about 1.5%, not more thanabout 1%, not more than about 0.75%, not more than about 0.5%, not morethan about 0.25%, not more than about 0.2% or not more than about 0.15%.In one such embodiment, free plasma EPA and/or total fatty acid levelsare determined prior to initiating therapy.

In another embodiment, the subject or subject group being treated inaccordance with methods of the invention exhibits a fasting baselineabsolute plasma level of total fatty acid (or mean thereof) not greaterthan about 250 nmol/ml, not greater than about 200 nmol/ml, not greaterthan about 150 nmol/ml, not greater than about 100 nmol/ml, or notgreater than about 50 nmol/ml.

In another embodiment, the subject or subject group being treated inaccordance with methods of the invention exhibits a fasting baselineplasma, serum or red blood cell membrane EPA level not greater thanabout 70 μg/ml, not greater than about 60 μg/ml, not greater than about50 μg/ml, not greater than about 40 μg/ml, not greater than about 30μg/ml, or not greater than about 25 μg/ml.

In another embodiment, methods of the present invention comprise a stepof measuring the subject's (or subject group's mean) baseline lipidprofile prior to initiating therapy. In another embodiment, methods ofthe invention comprise the step of identifying a subject or subjectgroup having one or more of the following: baseline non-HDL-C value ofabout 200 mg/dl to about 400 mg/dl, for example at least about 210mg/dl, at least about 220 mg/dl, at least about 230 mg/dl, at leastabout 240 mg/dl, at least about 250 mg/dl, at least about 260 mg/dl, atleast about 270 mg/dl, at least about 280 mg/dl, at least about 290mg/dl, or at least about 300 mg/dl; baseline total cholesterol value ofabout 250 mg/dl to about 400 mg/dl, for example at least about 260mg/dl, at least about 270 mg/dl, at least about 280 mg/dl or at leastabout 290 mg/dl; baseline vLDL-C value of about 140 mg/dl to about 200mg/dl, for example at least about 150 mg/dl, at least about 160 mg/dl,at least about 170 mg/dl, at least about 180 mg/dl or at least about 190mg/dl; baseline HDL-C value of about 10 to about 60 mg/dl, for examplenot more than about 40 mg/dl, not more than about 35 mg/dl, not morethan about 30 mg/dl, not more than about 25 mg/dl, not more than about20 mg/dl, or not more than about 15 mg/dl; and/or baseline LDL-C valueof about 50 to about 300 mg/dl, for example not less than about 100mg/dl, not less than about 90 mg/dl, not less than about 80 mg/dl, notless than about 70 mg/dl, not less than about 60 mg/dl or not less thanabout 50 mg/dl.

In a related embodiment, upon treatment in accordance with the presentinvention, for example over a period of about 1 to about 200 weeks,about 1 to about 100 weeks, about 1 to about 80 weeks, about 1 to about50 weeks, about 1 to about 40 weeks, about 1 to about 20 weeks, about 1to about 15 weeks, about 1 to about 12 weeks, about 1 to about 10 weeks,about 1 to about 5 weeks, about 1 to about 2 weeks or about 1 week, thesubject or subject group exhibits one or more of the following outcomes:

(a) reduced triglyceride levels compared to baseline or placebo control;

(b) reduced Apo B levels compared to baseline or placebo control;

(c) increased HDL-C levels compared to baseline or placebo control;

(d) no increase in LDL-C levels compared to baseline or placebo control;

(e) a reduction in LDL-C levels compared to baseline or placebo control;

(f) a reduction in non-HDL-C levels compared to baseline or placebocontrol;

(g) a reduction in vLDL levels compared to baseline or placebo control;

(h) an increase in apo A-I levels compared to baseline or placebocontrol;

(i) an increase in apo A-I/apo B ratio compared to baseline or placebocontrol;

(j) a reduction in lipoprotein A levels compared to baseline or placebocontrol;

(k) a reduction in LDL particle number compared to baseline or placebocontrol;

(l) an increase in LDL size compared to baseline or placebo control;

(m) a reduction in remnant-like particle cholesterol compared tobaseline or placebo control;

(n) a reduction in oxidized LDL compared to baseline or placebo control;

(o) no change or a reduction in fasting plasma glucose (FPG) compared tobaseline or placebo control;

(p) a reduction in hemoglobin A_(1c) (HbA_(1c)) compared to baseline orplacebo control;

(q) a reduction in homeostasis model insulin resistance compared tobaseline or placebo control;

(r) a reduction in lipoprotein associated phospholipase A2 compared tobaseline or placebo control;

(s) a reduction in intracellular adhesion molecule-1 compared tobaseline or placebo control;

(t) a reduction in interleukin-6 compared to baseline or placebocontrol;

(u) a reduction in plasminogen activator inhibitor-1 compared tobaseline or placebo control;

(v) a reduction in high sensitivity C-reactive protein (hsCRP) comparedto baseline or placebo control;

(w) an increase in serum or plasma EPA compared to baseline;

(x) an increase in red blood cell (RBC) membrane EPA compared tobaseline or placebo control;

(y) a reduction or increase in one or more of serum phospholipid and/orred blood cell content of docosahexaenoic acid (DHA), docosapentaenoicacid (DPA), arachidonic acid (AA), palmitic acid (PA), staeridonic acid(SA) or oleic acid (OA) compared to baseline or placebo control;

(z) a reduction in one or more protein components of VLDL such asapolipoprotein C-III (hereinafter “APOC3”; also referred to as APOCIIIor HALP2) compared to baseline or placebo control;

(aa) no significant increase in, or a reduction in HDL-APOC3, comparedto baseline or placebo control; and/or

(bb) no significant increase in, or a reduction in non-HDL-APOC3,compared to baseline or placebo control.

In one embodiment, upon administering a composition of the invention toa subject, the subject exhibits a decrease in triglyceride levels, anincrease in the concentrations of EPA and DPA (n−3) in red blood cells,and an increase of the ratio of EPA:arachidonic acid in red blood cells.In a related embodiment the subject exhibits substantially no or noincrease in RBC DHA.

In one embodiment, methods of the present invention comprise measuringbaseline levels of one or more markers set forth in (a)-(bb) above priorto dosing the subject or subject group. In another embodiment, themethods comprise administering a composition as disclosed herein to thesubject after baseline levels of one or more markers set forth in(a)-(bb) are determined, and subsequently taking an additionalmeasurement of said one or more markers.

In another embodiment, upon treatment with a composition of the presentinvention, for example over a period of about 1 to about 200 weeks,about 1 to about 100 weeks, about 1 to about 80 weeks, about 1 to about50 weeks, about 1 to about 40 weeks, about 1 to about 20 weeks, about 1to about 15 weeks, about 1 to about 12 weeks, about 1 to about 10 weeks,about 1 to about 5 weeks, about 1 to about 2 weeks or about 1 week, thesubject or subject group exhibits any 2 or more of, any 3 or more of,any 4 or more of, any 5 or more of, any 6 or more of, any 7 or more of,any 8 or more of, any 9 or more of, any 10 or more of, any 11 or moreof, any 12 or more of, any 13 or more of, any 14 or more of, any 15 ormore of, any 16 or more of, any 17 or more of, any 18 or more of, any 19or more of, any 20 or more of, any 21 or more of, any 22 or more of, any23 or more of, any 24 or more of, any 25 or more of, any 26 or more of,any 27 or more of, or all 28 of outcomes (a)-(bb) described immediatelyabove.

In another embodiment, upon treatment with a composition of the presentinvention, the subject or subject group exhibits one or more of thefollowing outcomes:

(a) a reduction in triglyceride level of at least about 5%, at leastabout 10%, at least about 15%, at least about 20%, at least about 25%,at least about 30%, at least about 35%, at least about 40%, at leastabout 45%, at least about 50%, at least about 55% or at least about 75%(actual % change or median % change) as compared to baseline or placebocontrol;

(b) a less than 30% increase, less than 20% increase, less than 10%increase, less than 5% increase or no increase in non-HDL-C levels or areduction in non-HDL-C levels of at least about 1%, at least about 3%,at least about 5%, at least about 10%, at least about 15%, at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 55% or at least about 75% (actual % change or median % change) ascompared to baseline or placebo control;

(c) substantially no change in HDL-C levels, no change in HDL-C levels,or an increase in HDL-C levels of at least about 5%, at least about 10%,at least about 15%, at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 55% or at least about 75% (actual %change or median % change) as compared to baseline or placebo control;

(d) a less than 60% increase, a less than 50% increase, a less than 40%increase, a less than 30% increase, less than 20% increase, less than10% increase, less than 5% increase or no increase in LDL-C levels or areduction in LDL-C levels of at least about 5%, at least about 10%, atleast about 15%, at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 55%, at least about 55% or at leastabout 75% (actual % change or median % change) as compared to baselineor placebo control;

(e) a decrease in Apo B levels of at least about 5%, at least about 10%,at least about 15%, at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 55% or at least about 75% (actual %change or median % change) as compared to baseline or placebo control;

(f) a reduction in vLDL levels of at least about 5%, at least about 10%,at least about 15%, at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, or at least about 100% (actual % change or median %change) compared to baseline or placebo control;

(g) an increase in apo A-I levels of at least about 5%, at least about10%, at least about 15%, at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, or at least about 100% (actual % change ormedian % change) compared to baseline or placebo control;

(h) an increase in apo A-I/apo B ratio of at least about 5%, at leastabout 10%, at least about 15%, at least about 20%, at least about 25%,at least about 30%, at least about 35%, at least about 40%, at leastabout 45%, at least about 50%, or at least about 100% (actual % changeor median % change) compared to baseline or placebo control;

(i) a reduction in lipoprotein (a) levels of at least about 5%, at leastabout 10%, at least about 15%, at least about 20%, at least about 25%,at least about 30%, at least about 35%, at least about 40%, at leastabout 45%, at least about 50%, or at least about 100% (actual % changeor median % change) compared to baseline;

(j) a reduction in mean LDL particle number of at least about 5%, atleast about 10%, at least about 15%, at least about 20%, at least about25%, at least about 30%, at least about 35%, at least about 40%, atleast about 45%, at least about 50%, or at least about 100% (actual %change or median % change) compared to baseline or placebo control;

(k) an increase in mean LDL particle size of at least about 5%, at leastabout 10%, at least about 15%, at least about 20%, at least about 25%,at least about 30%, at least about 35%, at least about 40%, at leastabout 45%, at least about 50%, or at least about 100% (actual % changeor median % change) compared to baseline or placebo control;

(l) a reduction in remnant-like particle cholesterol of at least about5%, at least about 10%, at least about 15%, at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, or at least about 100% (actual %change or median % change) compared to baseline or placebo control;

(m) a reduction in oxidized LDL of at least about 5%, at least about10%, at least about 15%, at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, or at least about 100% (actual % change ormedian % change) compared to baseline or placebo control;

(n) substantially no change, no significant change, or a reduction (e.g.in the case of a diabetic subject) in fasting plasma glucose (FPG) of atleast about 5%, at least about 10%, at least about 15%, at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, or at leastabout 100% (actual % change or median % change) compared to baseline orplacebo control;

(o) substantially no change, no significant change or a reduction inhemoglobin A_(1c) (HbA_(1c)) of at least about 5%, at least about 10%,at least about 15%, at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,or at least about 50% (actual % change or median % change) compared tobaseline or placebo control;

(p) a reduction in homeostasis model index insulin resistance of atleast about 5%, at least about 10%, at least about 15%, at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, or at leastabout 100% (actual % change or median % change) compared to baseline orplacebo control;

(q) a reduction in lipoprotein associated phospholipase A2 of at leastabout 5%, at least about 10%, at least about 15%, at least about 20%, atleast about 25%, at least about 30%, at least about 35%, at least about40%, at least about 45%, at least about 50%, or at least about 100%(actual % change or median % change) compared to baseline or placebocontrol;

(r) a reduction in intracellular adhesion molecule-1 of at least about5%, at least about 10%, at least about 15%, at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, or at least about 100% (actual %change or median % change) compared to baseline or placebo control;

(s) a reduction in interleukin-6 of at least about 5%, at least about10%, at least about 15%, at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, or at least about 100% (actual % change ormedian % change) compared to baseline or placebo control;

(t) a reduction in plasminogen activator inhibitor-1 of at least about5%, at least about 10%, at least about 15%, at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, or at least about 100% (actual %change or median % change) compared to baseline or placebo control;

(u) a reduction in high sensitivity C-reactive protein (hsCRP) of atleast about 5%, at least about 10%, at least about 15%, at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, or at leastabout 100% (actual % change or median % change) compared to baseline orplacebo control;

(v) an increase in serum, plasma and/or RBC EPA of at least about 5%, atleast about 10%, at least about 15%, at least about 20%, at least about25%, at least about 30%, at least about 35%, at least about 40%, atleast about 45%, at least about 50%, at least about 100%, at least about200% or at least about 400% (actual % change or median % change)compared to baseline or placebo control;

(w) an increase in serum phospholipid and/or red blood cell membrane EPAof at least about 5%, at least about 10%, at least about 15%, at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, r at least about 50%, at leastabout 100%, at least about 200%, or at least about 400% (actual % changeor median % change) compared to baseline or placebo control;

(x) a reduction or increase in one or more of serum phospholipid and/orred blood cell DHA, DPA, AA, PA and/or OA of at least about 5%, at leastabout 10%, at least about 15%, at least about 20%, at least about 25%,at least about 30%, at least about 35%, at least about 40%, at leastabout 45%, at least about 50%, at least about 55% or at least about 75%(actual % change or median % change) compared to baseline or placebocontrol;

(y) a reduction in total cholesterol of at least about 5%, at leastabout 10%, at least about 15%, at least about 20%, at least about 25%,at least about 30%, at least about 35%, at least about 40%, at leastabout 45%, at least about 50%, at least about 55% or at least about 75%(actual % change or median % change) compared to baseline or placebocontrol;

(z) no significant increase in, or a reduction in one or more proteincomponents of VLDL such as APOC3, of at least about 0.2%, at least about0.3%, at least about 0.5%, at least about 0.6%, at least about 1%, atleast about 5%, at least about 8%, at least about 8.5%, at least about9%, at least about 9.4%, at least about 10%, at least about 10.1%, atleast about 14.3%, at least about 15%, at least about 19%, at leastabout 19.2%, at least about 20%, at least about 25%, at least about25.1%, at least about 30%, at least about 35%, at least about 40%, atleast about 45%, at least about 50%, at least about 55%, at least about65%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about100%, at least about 105%, at least about 110%, at least about 115%, atleast about 120%, at least about 125%, at least about 130%, at leastabout 135%, at least about 140%, at least about 145%, at least about150%, at least about 155%, at least about 160%, at least about 165%, atleast about 170%, at least about 175%, at least about 180%, at leastabout 185%, at least about 190%, at least about 195%, or at least about200% (actual % change or median % change) compared to baseline orplacebo control;

(aa) no significant increase in, or a reduction in HDL-APOC3, of atleast about 0.2%, at least about 0.3%, at least about 0.5%, at leastabout 0.6%, at least about 1%, at least about 5%, at least about 5.9%,at least about 8%, at least about 9%, at least about 10%, at least about15%, at least about 20%, at least about 21%, at least about 21.4%, atleast about 23%, at least about 23.3%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 55%, at least about 65%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 95%, at least about 100%, at leastabout 105%, at least about 110%, at least about 115%, at least about120%, at least about 125%, at least about 130%, at least about 135%, atleast about 140%, at least about 145%, at least about 150%, at leastabout 155%, at least about 160%, at least about 165%, at least about170%, at least about 175%, at least about 180%, at least about 185%, atleast about 190%, at least about 195%, or at least about 200% (actual %change or median % change) compared to baseline or placebo control;and/or

(bb) no significant increase in, or a reduction in non-HDL-APOC3, of atleast about 0.2%, at least about 0.3%, at least about 0.5%, at leastabout 0.6%, at least about 1%, at least about 1.4%, at least about 5%,at least about 5.4%, at least about 8%, at least about 9%, at leastabout 10%, at least about 15%, at least about 20%, at least about 22%,at least about 22.5%, at least about 25%, at least about 30%, at leastabout 31%, at least about 31.8%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, at least about 55%, at leastabout 65%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 100%, at least about 105%, at least about 110%, at least about115%, at least about 120%, at least about 125%, at least about 130%, atleast about 135%, at least about 140%, at least about 145%, at leastabout 150%, at least about 155%, at least about 160%, at least about165%, at least about 170%, at least about 175%, at least about 180%, atleast about 185%, at least about 190%, at least about 195%, or at leastabout 200% (actual % change or median % change) compared to baseline orplacebo control.

In one embodiment, methods of the present invention comprise measuringbaseline levels of one or more markers set forth in (a)-(bb) prior todosing the subject or subject group. In another embodiment, the methodscomprise administering a composition as disclosed herein to the subjectafter baseline levels of one or more markers set forth in (a)-(bb) aredetermined, and subsequently taking a second measurement of the one ormore markers as measured at baseline for comparison thereto.

In another embodiment, upon treatment with a composition of the presentinvention, for example over a period of about 1 to about 200 weeks,about 1 to about 100 weeks, about 1 to about 80 weeks, about 1 to about50 weeks, about 1 to about 40 weeks, about 1 to about 20 weeks, about 1to about 15 weeks, about 1 to about 12 weeks, about 1 to about 10 weeks,about 1 to about 5 weeks, about 1 to about 2 weeks or about 1 week, thesubject or subject group exhibits any 2 or more of, any 3 or more of,any 4 or more of, any 5 or more of, any 6 or more of, any 7 or more of,any 8 or more of, any 9 or more of, any 10 or more of, any 11 or moreof, any 12 or more of, any 13 or more of, any 14 or more of, any 15 ormore of, any 16 or more of, any 17 or more of, any 18 or more of, any 19or more of, any 20 or more of, any 21 or more of, any 22 or more of, any23 or more of, any 24 or more of, any 25 or more of, any 26 or more of,any 27 or more of, or all 28 of outcomes (a)-(bb) described immediatelyabove.

Parameters (a)-(bb) can be measured in accordance with any clinicallyacceptable methodology. For example, triglycerides, total cholesterol,HDL-C and fasting blood sugar can be sample from serum and analyzedusing standard photometry techniques. VLDL-TG, LDL-C and VLDL-C can becalculated or determined using serum lipoprotein fractionation bypreparative ultracentrifugation and subsequent quantitative analysis byrefractometry or by analytic ultracentrifugal methodology. Apo A1, Apo Band hsCRP can be determined from serum using standard nephelometrytechniques. Lipoprotein (a) can be determined from serum using standardturbidimetric immunoassay techniques. LDL particle number and particlesize can be determined using nuclear magnetic resonance (NMR)spectrometry. Remnants lipoproteins and LDL-phospholipase A2 can bedetermined from EDTA plasma or serum and serum, respectively, usingenzymatic immunoseparation techniques. Oxidized LDL, intercellularadhesion molecule-1 and interleukin-6 levels can be determined fromserum using standard enzyme immunoassay techniques. APOC3 levels can bedetermined by known quantitative methods including, for example,antibody-based assays such as ELISA. These techniques are described indetail in standard textbooks, for example Tietz Fundamentals of ClinicalChemistry, 6th Ed. (Burtis, Ashwood and Borter Eds.), WB SaundersCompany.

In one embodiment, the present invention provides a method of reducingan APOC3 level of a subject having baseline fasting triglycerides ofabout 200 mg/dl to about 499 mg/dl and on statin therapy (e.g., stablestatin therapy), the method comprising administering to the subject apharmaceutical composition comprising about 1 g to about 4 g of EPA perday, wherein upon administering the composition to the subject daily fora period of 1 to about 12 weeks, the subject exhibits a decrease inAPOC3 of at least about 5% compared to a control subject on stablestatin therapy without EPA, wherein the control subject also hasbaseline fasting triglycerides of about 200 mg/dl to about 499 mg/dl. Insome embodiments, the decrease in APOC3 level is at least about 7.6%, atleast about 10%, at least about 13.1%, at least about 25%, at leastabout 50%, at least about 75%, at least about 100%, at least about 125%,at least about 150%, at least about 175%, or at least about 200%. Insome embodiments, the subject is administered about 2 g per day of EPA.In some embodiments, the subject is administered about 3.8 g to about 4g per day of EPA. In any of the methods described herein, the EPA can beethyl eicosapentaenoate. In some embodiments, the subject exhibits adecrease in HDL-ApoC3 of at least about 5%, at least about 10%, at leastabout 15%, at least about 20%, at least about 21%, about 21.4%, or atleast about 25% compared to baseline. In some embodiments, the subjectexhibits a decrease in HDL-ApoC3 of at least about 5%, at least about10%, at least about 15%, at least about 20%, at least about 23%, about23.3%, at least about 25% compared to the control subject. In someembodiments, the subject exhibits a decrease in non-HDL-ApoC3 of atleast about 5%, about 5.4%, at least about 10%, at least about 15%, orat least about 20% compared to baseline. In some embodiments, thesubject exhibits a decrease in non-HDL-ApoC3 of at least about 5%, atleast about 10%, at least about 15%, at least about 20%, at least about22%, about 22.5%, at least about 25%, at least about 30%, at least about31%, about 31.8%, or at least about 35% compared to the control subject.

Apolipoprotein C-III (APOC3, APOCIII or HALP2) is a 9-kDa proteincomponent of VLDL. It is synthesized in the liver and is known toinhibit lipoprotein lipase and hepatic lipase, and is additionallybelieved to inhibit hepatic uptake of triglyceride-rich particles bymodulating the binding of chylomicron remnants and VLDL to receptors.Accordingly, APOC3 is thought to inhibit the clearance of triglyceridesfrom the blood. APOC3 is typically glycosylated; at least threepolymorphic forms with varying amounts of sialic acid have beenidentified (Apo C-III-0, Apo C-III-1, and Apo C-III-2). Clinically,subjects with normal or non-insulin diabetes mellitus who have certainhaplotypes in the ApoAI-CIII-AIV gene cluster on chromosome 11q23 havean approximately threefold higher risk of coronary heart disease. Peoplewho do not produce APOC3 have lower levels of triglycerides and lowerinstances of cardiovascular disease.

In one embodiment, the present invention provides a method of reducingan APOC3 level of a subject having baseline fasting triglycerides ofabout 500 mg/dl to about 1500 mg/dl, the method comprising administeringto the subject a pharmaceutical composition comprising about 1 g toabout 4 g of EPA per day, wherein upon administering the composition tothe subject daily for a period of 1 to about 12 weeks, the subjectexhibits a decrease in APOC3 in plasma and/or RBCs of at least about 5%compared to a control subject who has not received about 1 g to about 4g of EPA per day, wherein the control subject also has baseline fastingtriglycerides of about 500 mg/dl to about 1500 mg/dl. In someembodiments, the decrease in APOC3 level is at least about 10%, at leastabout 11%, at least about 14%, at least about 25%, or at least about50%, at least about 75%, at least about 100%, at least about 125%, atleast about 150%, at least about 175%, or at least about 200%. In someembodiments, the subject is administered about 2 g per day of EPA. Insome embodiments, the subject is administered about 3.8 g to about 4 gper day of EPA. In any of the methods described herein, the EPA can beethyl eicosapentaenoate. In some embodiments, the subject exhibits adecrease in HDL-ApoC3 of at least about 5% compared to baseline.

In one embodiment, subjects fast for up to 12 hours prior to bloodsample collection, for example about 10 hours.

In another embodiment, the present invention provides a method oftreating or preventing primary hypercholesterolemia and/or mixeddyslipidemia (Fredrickson Types IIa and IIb) in a patient in needthereof, comprising administering to the patient one or morecompositions as disclosed herein. In a related embodiment, the presentinvention provides a method of reducing triglyceride levels in a subjector subjects when treatment with a statin or niacin extended-releasemonotherapy is considered inadequate (Frederickson type IVhyperlipidemia).

In another embodiment, the present invention provides a method oftreating or preventing risk of recurrent nonfatal myocardial infarctionin a patient with a history of myocardial infarction, comprisingadministering to the patient one or more compositions as disclosedherein.

In another embodiment, the present invention provides a method ofslowing progression of or promoting regression of atheroscleroticdisease in a patient in need thereof, comprising administering to asubject in need thereof one or more compositions as disclosed herein.

In another embodiment, the present invention provides a method oftreating or preventing very high serum triglyceride levels (e.g. TypesIV and V hyperlipidemia) in a patient in need thereof, comprisingadministering to the patient one or more compositions as disclosedherein.

In another embodiment, the present invention provides a method oftreating subjects having very high serum triglyceride levels (e.g.greater than 1000 mg/dl or greater than 2000 mg/dl) and that are at riskof developing pancreatitis, comprising administering to the patient oneor more compositions as disclosed herein.

In one embodiment, a composition of the invention is administered to asubject in an amount sufficient to provide a daily dose ofeicosapentaenoic acid of about 1 mg to about 10,000 mg, 25 about 5000mg, about 50 to about 3000 mg, about 75 mg to about 2500 mg, about 2 gto about 4 g, about 3.8 g to about 4 g, or about 100 mg to about 1000mg, for example about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg,about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about70 mg, about 75 mg, about 100 mg, about 125 mg, about 150 mg, about 175mg, about 200 mg, about 225 mg, about 250 mg, about 275 mg, about 300mg, about 325 mg, about 350 mg, about 375 mg, about 400 mg, about 425mg, about 450 mg, about 475 mg, about 500 mg, about 525 mg, about 550mg, about 575 mg, about 600 mg, about 625 mg, about 650 mg, about 675mg, about 700 mg, about 725 mg, about 750 mg, about 775 mg, about 800mg, about 825 mg, about 850 mg, about 875 mg, about 900 mg, about 925mg, about 950 mg, about 975 mg, about 1000 mg, about 1025 mg, about 1050mg, about 1075 mg, about 1100 mg, about 1025 mg, about 1050 mg, about1075 mg, about 1200 mg, about 1225 mg, about 1250 mg, about 1275 mg,about 1300 mg, about 1325 mg, about 1350 mg, about 1375 mg, about 1400mg, about 1425 mg, about 1450 mg, about 1475 mg, about 1500 mg, about1525 mg, about 1550 mg, about 1575 mg, about 1600 mg, about 1625 mg,about 1650 mg, about 1675 mg, about 1700 mg, about 1725 mg, about 1750mg, about 1775 mg, about 1800 mg, about 1825 mg, about 1850 mg, about1875 mg, about 1900 mg, about 1925 mg, about 1950 mg, about 1975 mg,about 2000 mg, about 2025 mg, about 2050 mg, about 2075 mg, about 2100mg, about 2125 mg, about 2150 mg, about 2175 mg, about 2200 mg, about2225 mg, about 2250 mg, about 2275 mg, about 2300 mg, about 2325 mg,about 2350 mg, about 2375 mg, about 2400 mg, about 2425 mg, about 2450mg, about 2475 mg, about 2500 mg, about 2750 mg, about 3000 mg, about3250 mg, about 3500 mg, about 3750 mg, about 3800 mg, about 4000 mg,about 4250 mg, about 4500 mg, about 4750 mg, about 5000 mg, about 5500mg, about 6000 mg, about 6500 mg, about 7000 mg, about 7500 mg, about8000 mg, about 8500 mg, about 9000 mg, about 9500 mg, or about 10,000mg.

In another embodiment, any of the methods disclosed herein are used intreatment or prevention of a subject or subjects that consume atraditional Western diet. In one embodiment, the methods of theinvention include a step of identifying a subject as a Western dietconsumer or prudent diet consumer and then treating the subject if thesubject is deemed a Western diet consumer. The term “Western diet”herein refers generally to a typical diet consisting of, by percentageof total calories, about 45% to about 50% carbohydrate, about 35% toabout 40% fat, and about 10% to about 15% protein. A Western diet mayalternately or additionally be characterized by relatively high intakesof red and processed meats, sweets, refined grains, and desserts, forexample more than 50%, more than 60% or more or 70% of total caloriescome from these sources.

In one embodiment, a composition for use in methods of the inventioncomprises eicosapentaenoic acid, or a pharmaceutically acceptable ester,derivative, conjugate or salt thereof, or mixtures of any of theforegoing, collectively referred to herein as “EPA.” The term“pharmaceutically acceptable” in the present context means that thesubstance in question does not produce unacceptable toxicity to thesubject or interaction with other components of the composition.

In one embodiment, the EPA comprises all-ciseicosa-5,8,11,14,17-pentaenoic acid. In another embodiment, the EPAcomprises an eicosapentaenoic acid ester. In another embodiment, the EPAcomprises a C₁-C₅ alkyl ester of eicosapentaenoic acid. In anotherembodiment, the EPA comprises eicosapentaenoic acid ethyl ester,eicosapentaenoic acid methyl ester, eicosapentaenoic acid propyl ester,or eicosapentaenoic acid butyl ester. In another embodiment, the EPAcomprises In one embodiment, the EPA comprises all-ciseicosa-5,8,11,14,17-pentaenoic acid ethyl ester.

In another embodiment, the EPA is in the form of ethyl-EPA, lithium EPA,mono-, di- or triglyceride EPA or any other ester or salt of EPA, or thefree acid form of EPA. The EPA may also be in the form of a2-substituted derivative or other derivative which slows down its rateof oxidation but does not otherwise change its biological action to anysubstantial degree.

In another embodiment, EPA is present in a composition useful inaccordance with methods of the invention in an amount of about 50 mg toabout 5000 mg, about 75 mg to about 2500 mg, or about 100 mg to about1000 mg, for example about 50 mg, about 75 mg, about 100 mg, about 125mg, about 150 mg, about 175 mg, about 200 mg, about 225 mg, about 250mg, about 275 mg, about 300 mg, about 325 mg, about 350 mg, about 375mg, about 400 mg, about 425 mg, about 450 mg, about 475 mg, about 500mg, about 525 mg, about 550 mg, about 575 mg, about 600 mg, about 625mg, about 650 mg, about 675 mg, about 700 mg, about 725 mg, about 750mg, about 775 mg, about 800 mg, about 825 mg, about 850 mg, about 875mg, about 900 mg, about 925 mg, about 950 mg, about 975 mg, about 1000mg, about 1025 mg, about 1050 mg, about 1075 mg, about 1100 mg, about1025 mg, about 1050 mg, about 1075 mg, about 1200 mg, about 1225 mg,about 1250 mg, about 1275 mg, about 1300 mg, about 1325 mg, about 1350mg, about 1375 mg, about 1400 mg, about 1425 mg, about 1450 mg, about1475 mg, about 1500 mg, about 1525 mg, about 1550 mg, about 1575 mg,about 1600 mg, about 1625 mg, about 1650 mg, about 1675 mg, about 1700mg, about 1725 mg, about 1750 mg, about 1775 mg, about 1800 mg, about1825 mg, about 1850 mg, about 1875 mg, about 1900 mg, about 1925 mg,about 1950 mg, about 1975 mg, about 2000 mg, about 2025 mg, about 2050mg, about 2075 mg, about 2100 mg, about 2125 mg, about 2150 mg, about2175 mg, about 2200 mg, about 2225 mg, about 2250 mg, about 2275 mg,about 2300 mg, about 2325 mg, about 2350 mg, about 2375 mg, about 2400mg, about 2425 mg, about 2450 mg, about 2475 mg, about 2500 mg, 2525 mg,about 2550 mg, about 2575 mg, about 2600 mg, about 2625 mg, about 2650mg, about 2675 mg, about 2700 mg, about 2725 mg, about 2750 mg, about2775 mg, about 2800 mg, about 2825 mg, about 2850 mg, about 2875 mg,about 2900 mg, about 2925 mg, about 2950 mg, about 2975 mg, about 3000mg, about 3025 mg, about 3050 mg, about 3075 mg, about 3100 mg, about3125 mg, about 3150 mg, about 3175 mg, about 3200 mg, about 3225 mg,about 3250 mg, about 3275 mg, about 3300 mg, about 3325 mg, about 3350mg, about 3375 mg, about 3400 mg, about 3425 mg, about 3450 mg, about3475 mg, about 3500 mg, about 3525 mg, about 3550 mg, about 3575 mg,about 3600 mg, about 3625 mg, about 3650 mg, about 3675 mg, about 3700mg, about 3725 mg, about 3750 mg, about 3775 mg, about 3800 mg, about3825 mg, about 3850 mg, about 3875 mg, about 3900 mg, about 3925 mg,about 3950 mg, about 3975 mg, about 4000 mg, about 4025 mg, about 4050mg, about 4075 mg, about 4100 mg, about 4125 mg, about 4150 mg, about4175 mg, about 4200 mg, about 4225 mg, about 4250 mg, about 4275 mg,about 4300 mg, about 4325 mg, about 4350 mg, about 4375 mg, about 4400mg, about 4425 mg, about 4450 mg, about 4475 mg, about 4500 mg, about4525 mg, about 4550 mg, about 4575 mg, about 4600 mg, about 4625 mg,about 4650 mg, about 4675 mg, about 4700 mg, about 4725 mg, about 4750mg, about 4775 mg, about 4800 mg, about 4825 mg, about 4850 mg, about4875 mg, about 4900 mg, about 4925 mg, about 4950 mg, about 4975 mg, orabout 5000 mg.

In another embodiment, a composition useful in accordance with theinvention contains not more than about 10%, not more than about 9%, notmore than about 8%, not more than about 7%, not more than about 6%, notmore than about 5%, not more than about 4%, not more than about 3%, notmore than about 2%, not more than about 1%, or not more than about 0.5%,by weight, docosahexaenoic acid (DHA), if any. In another embodiment, acomposition of the invention contains substantially no docosahexaenoicacid. In still another embodiment, a composition useful in the presentinvention contains no docosahexaenoic acid and/or derivative thereof.

In another embodiment, EPA comprises at least 70%, at least 80%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100%, by weight, of all fatty acids present in acomposition that is useful in methods of the present invention.

In one embodiment, a composition of the invention comprises ultra-pureEPA. The term “ultra-pure” as used herein with respect to EPA refers toa composition comprising at least 95% by weight EPA (as the term “EPA”is defined and exemplified herein). Ultra-pure EPA comprises at least96% by weight EPA, at least 97% by weight EPA, or at least 98% by weightEPA, wherein the EPA is any form of EPA as set forth herein.

In another embodiment, a composition useful in accordance with methodsof the invention contains less than 10%, less than 9%, less than 8%,less than 7%, less than 6%, less than 5%, less than 4%, less than 3%,less than 2%, less than 1%, less than 0.5% or less than 0.25%, by weightof the total composition or by weight of the total fatty acid content,of any fatty acid other than EPA. Illustrative examples of a “fatty acidother than EPA” include linolenic acid (LA), arachidonic acid (AA),docosahexaenoic acid (DHA), alpha-linolenic acid (ALA), stearadonic acid(STA), eicosatrienoic acid (ETA) and/or docosapentaenoic acid (DPA). Inanother embodiment, a composition useful in accordance with methods ofthe invention contains about 0.1% to about 4%, about 0.5% to about 3%,or about 1% to about 2%, by weight, of total fatty acids other than EPAand/or DHA.

In another embodiment, a composition useful in accordance with theinvention has one or more of the following features: (a)eicosapentaenoic acid ethyl ester represents at least about 96%, atleast about 97%, or at least about 98%, by weight, of all fatty acidspresent in the composition; (b) the composition contains not more thanabout 4%, not more than about 3%, or not more than about 2%, by weight,of total fatty acids other than eicosapentaenoic acid ethyl ester; (c)the composition contains not more than about 0.6%, not more than about0.5%, or not more than about 0.4% of any individual fatty acid otherthan eicosapentaenoic acid ethyl ester; (d) the composition has arefractive index (20° C.) of about 1 to about 2, about 1.2 to about 1.8or about 1.4 to about 1.5; (e) the composition has a specific gravity(20° C.) of about 0.8 to about 1.0, about 0.85 to about 0.95 or about0.9 to about 0.92; (e) the composition contains not more than about 20ppm, not more than about 15 ppm or not more than about 10 ppm heavymetals, (f) the composition contains not more than about 5 ppm, not morethan about 4 ppm, not more than about 3 ppm, or not more than about 2ppm arsenic, and/or (g) the composition has a peroxide value of not morethan about 5 meq/kg, not more than about 4 meq/kg, not more than about 3meq/kg, or not more than about 2 meq/kg.

In another embodiment, a composition useful in accordance with theinvention comprises, consists of or consists essentially of at least 95%by weight ethyl eicosapentaenoate (EPA-E), about 0.2% to about 0.5% byweight ethyl octadecatetraenoate (ODTA-E), about 0.05% to about 0.25% byweight ethyl nonaecapentaenoate (NDPA-E), about 0.2% to about 0.45% byweight ethyl arachidonate (AA-E), about 0.3% to about 0.5% by weightethyl eicosatetraenoate (ETA-E), and about 0.05% to about 0.32% ethylheneicosapentaenoate (HPA-E). In another embodiment, the composition ispresent in a capsule shell.

In another embodiment, compositions useful in accordance with theinvention comprise, consist essential of, or consist of at least 95%,96% or 97%, by weight, ethyl eicosapentaenoate, about 0.2% to about 0.5%by weight ethyl octadecatetraenoate, about 0.05% to about 0.25% byweight ethyl nonaecapentaenoate, about 0.2% to about 0.45% by weightethyl arachidonate, about 0.3% to about 0.5% by weight ethyleicosatetraenoate, and about 0.05% to about 0.32% ethylheneicosapentaenoate. Optionally, the composition contains not more thanabout 0.06%, about 0.05%, or about 0.04%, by weight, DHA or derivativethereof such as ethyl-DHA. In one embodiment the composition containssubstantially no or no amount of DHA or derivative thereof such asethyl-DHA. The composition further optionally comprises one or moreantioxidants (e.g. tocopherol) or other impurities in an amount of notmore than about 0.5% or not more than 0.05%. In another embodiment, thecomposition comprises about 0.05% to about 0.4%, for example about 0.2%by weight tocopherol. In another embodiment, about 500 mg to about 1 gof the composition is provided in a capsule shell.

In another embodiment, compositions useful in accordance with theinvention comprise, consist essential of, or consist of at least 96% byweight ethyl eicosapentaenoate, about 0.22% to about 0.4% by weightethyl octadecatetraenoate, about 0.075% to about 0.20% by weight ethylnonaecapentaenoate, about 0.25% to about 0.40% by weight ethylarachidonate, about 0.3% to about 0.4% by weight ethyl eicosatetraenoateand about 0.075% to about 0.25% ethyl heneicosapentaenoate. Optionally,the composition contains not more than about 0.06%, about 0.05%, orabout 0.04%, by weight, DHA or derivative thereof such as ethyl-DHA. Inone embodiment the composition contains substantially no or no amount ofDHA or derivative thereof such as ethyl-DHA. The composition furtheroptionally comprises one or more antioxidants (e.g. tocopherol) or otherimpurities in an amount of not more than about 0.5% or not more than0.05%. In another embodiment, the composition comprises about 0.05% toabout 0.4%, for example about 0.2% by weight tocopherol. In anotherembodiment, the invention provides a dosage form comprising about 500 mgto about 1 g of the foregoing composition in a capsule shell. In oneembodiment, the dosage form is a gel or liquid capsule and is packagedin blister packages of about 1 to about 20 capsules per sheet.

In another embodiment, compositions useful in accordance with theinvention comprise, consist essential of, or consist of at least 96%,97% or 98%, by weight, ethyl eicosapentaenoate, about 0.25% to about0.38% by weight ethyl octadecatetraenoate, about 0.10% to about 0.15% byweight ethyl nonaecapentaenoate, about 0.25% to about 0.35% by weightethyl arachidonate, about 0.31% to about 0.38% by weight ethyleicosatetraenoate, and about 0.08% to about 0.20% ethylheneicosapentaenoate. Optionally, the composition contains not more thanabout 0.06%, about 0.05%, or about 0.04%, by weight, DHA or derivativethereof such as ethyl-DHA. In one embodiment the composition containssubstantially no or no amount of DHA or derivative thereof such asethyl-DHA. The composition further optionally comprises one or moreantioxidants (e.g. tocopherol) or other impurities in an amount of notmore than about 0.5% or not more than 0.05%. In another embodiment, thecomposition comprises about 0.05% to about 0.4%, for example about 0.2%by weight tocopherol. In another embodiment, the invention provides adosage form comprising about 500 mg to about 1 g of the foregoingcomposition in a capsule shell.

In another embodiment, a composition as described herein is administeredto a subject once or twice per day. In another embodiment, 1, 2, 3 or 4capsules, each containing about 1 g of a composition as describedherein, are administered to a subject daily. In another embodiment, 1 or2 capsules, each containing about 1 g of a composition as describedherein, are administered to the subject in the morning, for examplebetween about 5 am and about 11 am, and 1 or 2 capsules, each containingabout 1 g of a composition as described herein, are administered to thesubject in the evening, for example between about 5 pm and about 11 pm.

In one embodiment, a subject being treated in accordance with methods ofthe invention is not otherwise on lipid-altering therapy, for examplestatin, fibrate, niacin and/or ezetimibe therapy.

In another embodiment, compositions useful in accordance with methods ofthe invention are orally deliverable. The terms “orally deliverable” or“oral administration” herein include any form of delivery of atherapeutic agent or a composition thereof to a subject wherein theagent or composition is placed in the mouth of the subject, whether ornot the agent or composition is swallowed. Thus “oral administration”includes buccal and sublingual as well as esophageal administration. Inone embodiment, the composition is present in a capsule, for example asoft gelatin capsule.

A composition for use in accordance with the invention can be formulatedas one or more dosage units. The terms “dose unit” and “dosage unit”herein refer to a portion of a pharmaceutical composition that containsan amount of a therapeutic agent suitable for a single administration toprovide a therapeutic effect. Such dosage units may be administered oneto a plurality (i.e. 1 to about 10, 1 to 8, 1 to 6, 1 to 4 or 1 to 2) oftimes per day, or as many times as needed to elicit a therapeuticresponse.

In another embodiment, the invention provides use of any compositiondescribed herein for treating moderate to severe hypertriglyceridemia ina subject in need thereof, comprising: providing a subject having afasting baseline triglyceride level of about 500 mg/dl to about 1500mg/dl and administering to the subject a pharmaceutical composition asdescribed herein. In one embodiment, the composition comprises about 1 gto about 4 g of eicosapentaenoic acid ethyl ester, wherein thecomposition contains substantially no docosahexaenoic acid.

In one embodiment, compositions of the invention, upon storage in aclosed container maintained at room temperature, refrigerated (e.g.about 5 to about 5-10° C.) temperature, or frozen for a period of about1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, exhibit at least about90%, at least about 95%, at least about 97.5%, or at least about 99% ofthe active ingredient(s) originally present therein.

In one embodiment, the invention provides use of a composition asdescribed herein in manufacture of a medicament for treatment of any ofa cardiovascular-related disease. In another embodiment, the subject isdiabetic.

In one embodiment, a composition as set forth herein is packagedtogether with instructions for using the composition to treat acardiovascular disorder.

EXAMPLES Example 1 Reduction of ApoC3 Levels in Subjects with BaselineFasting Triglyceride Levels of ≧500 mg/dl

A multi-center, placebo-controlled randomized, double-blind, 12-weekstudy with an open-label extension was performed to evaluate theefficacy and safety of AMR101 in patients with fasting triglyceridelevels≧500 mg/dL. The primary objective of the study was to determinethe efficacy of AMR101 2 g daily and 4 g daily, compared to placebo, inlowering fasting TG levels in patients with fasting TG levels≧500 mg/dLand ≦1500 mg/dL (≧5.65 mmol/L and ≦16.94 mmol/L).

The secondary objectives of this study were the following:

-   1. To determine the safety and tolerability of AMR101 2 g daily and    4 g daily;-   2. To determine the effect of AMR101 on lipid and apolipoprotein    profiles;-   3. To determine the effect of AMR101 on low-density lipoprotein    (LDL) particle number and size;-   4. To determine the effect of AMR101 on oxidized LDL;-   5. To determine the effect of AMR101 on fasting plasma glucose (FPG)    and hemoglobin A_(1c) (HbA_(1c));-   6. To determine the effect of AMR101 on insulin resistance;-   7. To determine the effect of AMR101 on high-sensitivity C-reactive    protein (hsCRP);-   8. To determine the effects of AMR101 2 g daily and 4 g daily on the    incorporation of fatty acids into red blood cell membranes and into    plasma phospholipids;-   9. To explore the relationship between baseline fasting TG levels    and the reduction in fasting TG levels; and-   10. To explore the relationship between an increase in red blood    cell membrane eicosapentaenoic acid (EPA) concentrations and the    reduction in fasting TG levels.

The population for this study was men and women (women of childbearingpotential needed to be on contraception or practice abstinence)>18 yearsof age with a body mass index≦45 kg/m² who were not on lipid-alteringtherapy or were not currently on lipid-altering therapy. Patientscurrently on statin therapy (with or without ezetimibe) were evaluatedby the investigator as to whether this therapy could be safelydiscontinued at screening, or if it should have been continued. Ifstatin therapy (with or without ezetimibe) was to be continued, dose(s)must have been stable for weeks prior to randomization. Patients takingnon-statin, lipid-altering medications (niacin>200 mg/day, fibrates,fish oil, other products containing omega-3 fatty acids, or other herbalproducts or dietary supplements with potential lipid-altering effects),either alone or in combination with statin therapy (with or withoutezetimibe), must have been able to safely discontinue non-statin,lipid-altering therapy at screening.

Approximately 240 patients were randomized at approximately 50 centersin North America, South America, Central America, Europe, India, andSouth Africa. The study was a 58- to 60-week, Phase 3, multi-centerstudy consisting of 3 study periods: (1) a 6- to 8-week screening periodthat included a diet and lifestyle stabilization and washout period anda TG qualifying period; (2) a 12-week, double-blind, randomized,placebo-controlled treatment period; and (3) a 40-week, open-label,extension period.

During the screening period and double-blind treatment period, allvisits were within ±3 days of the scheduled time. During the open-labelextension period, all visits were within ±7 days of the scheduled time.The screening period included a 4- or 6-week diet and lifestylestabilization period and washout period followed by a 2-week TGqualifying period.

The screening visit (Visit 1) occurred for all patients at either 6weeks (for patients not on lipid-altering therapy at screening or forpatients who did not need to discontinue their current lipid-alteringtherapy) or 8 weeks (for patients who required washout of their currentlipid-altering therapy at screening) before randomization, as follows:

Patients who did not require a washout: The screening visit will occurat Visit 1 (Week −6). Eligible patients entered a 4-week diet andlifestyle stabilization period. At the screening visit, all patientsreceived counseling regarding the importance of the National CholesterolEducation Program (NCEP) Therapeutic Lifestyle Changes (TLC) diet andreceived instructions on how to follow this diet.

Patients who required a washout: The screening visit occurred at Visit 1(Week −8). Eligible patients began a 6-week washout period at thescreening visit. Patients received counseling regarding the NCEP TLCdiet and received instructions on how to follow this diet. Sitepersonnel contacted patients who did not qualify for participation basedon screening laboratory test results to instruct them to resume theirprior lipid-altering medications.

At the end of the 4-week diet and lifestyle stabilization period or the6-week diet and stabilization and washout period, eligible patientsentered the 2-week TG qualifying period and had their fasting TG levelmeasured at Visit 2 (Week −2) and Visit 3 (Week −1). Eligible patientsmust have had an average fasting TG level≦500 mg/dL and ≦1500 mg/dL(≧5.65 mmol/L and ≦16.94 mmol/L) to enter the 12-week double-blindtreatment period. The TG level for qualification was based on theaverage (arithmetic mean) of the Visit 2 (Week −2) and Visit 3 (Week −1)values. If a patient's average TG level from Visit 2 and Visit 3 felloutside the required range for entry into the study, an additionalsample for fasting TG measurement was collected 1 week later at Visit3.1. If a third sample was collected at Visit 3.1, entry into the studywas based on the average (arithmetic mean) of the values from Visit 3and Visit 3.1.

After confirmation of qualifying fasting TG values, eligible patientsentered a 12-week, randomized, double-blind treatment period. At Visit 4(Week 0), patients were randomly assigned to one of the followingtreatment groups:

-   -   AMR101 2 g daily,    -   AMR101 4 g daily, or    -   Placebo.

During the double-blind treatment period, patients returned to the siteat Visit 5 (Week 4), Visit 6 (Week 11), and Visit 7 (Week 12) forefficacy and safety evaluations.

Patients who completed the 12-week double-blind treatment period wereeligible to enter a 40-week, open-label, extension period at Visit 7(Week 12). All patients received open-label AMR101 4 g daily. From Visit8 (Week 16) until the end of the study, changes to the lipid-alteringregimen were permitted (e.g., initiating or raising the dose of statinor adding non-statin, lipid-altering medications to the regimen), asguided by standard practice and prescribing information. After Visit 8(Week 16), patients returned to the site every 12 weeks until the lastvisit at Visit 11 (Week 52).

Eligible patients were randomly assigned at Visit 4 (Week 0) to orallyreceive AMR101 2 g daily, AMR101 4 g daily, or placebo for the 12-weekdouble-blind treatment period. AMR101 was provided in 1 g liquid-filled,oblong, gelatin capsules. The matching placebo capsule was filled withlight liquid paraffin and contained 0 g of AMR101. During thedouble-blind treatment period, patients took 2 capsules (AMR101 ormatching placebo) in the morning and 2 in the evening for a total of 4capsules per day. Patients in the AMR101 2 g/day treatment groupreceived 1 AMR101 1 g capsule and 1 matching placebo capsule in themorning and in the evening. Patients in the AMR101 4 g/day treatmentgroup received 2 AMR101 1 g capsules in the morning and evening.

Patients in the placebo group received 2 matching placebo capsules inthe morning and evening. During the extension period, patients receivedopen-label AMR101 4 g daily. Patients took 2 AMR101 1 g capsules in themorning and 2 in the evening.

The primary efficacy variable for the double-blind treatment period waspercent change in TG from baseline to Week 12 endpoint. The secondaryefficacy variables for the double-blind treatment period included thefollowing:

-   -   Percent changes in total cholesterol (TC), high-density        lipoprotein cholesterol (HDL-C), calculated low-density        lipoprotein cholesterol (LDL-C), calculated non-high-density        lipoprotein cholesterol (non-HDL-C), and very low-density        lipoprotein cholesterol (VLDL-C) from baseline to Week 12        endpoint;    -   Percent change in very low-density lipoprotein TG from baseline        to Week 12;    -   Percent changes in apolipoprotein A-I (apo A-I), apolipoprotein        B (apo B), and apo A-I/apo B ratio from baseline to Week 12;    -   Percent changes in lipoprotein (a) from baseline to Week 12        (selected sites only);    -   Percent changes in LDL particle number and size, measured by        nuclear magnetic resonance, from baseline to Week 12 (selected        sites only);    -   Percent change in remnant-like particle cholesterol from        baseline to Week 12 (selected sites only);    -   Percent change in oxidized LDL from baseline to Week 12        (selected sites only);    -   Changes in FPG and HbA_(1c) from baseline to Week 12;    -   Change in insulin resistance, as assessed by the homeostasis        model index insulin resistance, from baseline to Week 12;    -   Percent change in lipoprotein associated phospholipase A2 from        baseline to Week 12 (selected sites only);    -   Change in intracellular adhesion molecule-1 from baseline to        Week 12 (selected sites only);    -   Change in interleukin-6 from baseline to Week 12 (selected sites        only);    -   Change in plasminogen activator inhibitor-1 from baseline to        Week 12 (selected sites only);    -   Change in hsCRP from baseline to Week 12 (selected sites only);    -   Change in serum phospholipid EPA content from baseline to Week        12;    -   Change in red blood cell membrane EPA content from baseline to        Week 12; and    -   Change in serum phospholipid and red blood cell membrane content        in the following fatty acids from baseline to Week 12:        docosapentaenoic acid, docosahexaenoic acid, arachidonic acid,        palmitic acid, stearic acid, and oleic acid.

The efficacy variable for the open-label extension period was percentchange in fasting TG from extension baseline to end of treatment. Safetyassessments included adverse events, clinical laboratory measurements(chemistry, hematology, and urinalysis), 12-lead electrocardiograms(ECGs), vital signs, and physical examinations

For TG, TC, HDL-C, calculated LDL-C, calculated non-HDL-C, and VLDL-C,baseline was defined as the average of Visit 4 (Week 0) and thepreceding lipid qualifying visit (either Visit 3 [Week −1] or if itoccurs, Visit 3.1) measurements. Baseline for all other efficacyparameters was the Visit 4 (Week 0) measurement.

For TC, HDL-C, calculated LDL-C, calculated non-HDL-C, and VLDL-C, Week12 endpoint was defined as the average of Visit 6 (Week 11) and Visit 7(Week 12) measurements. Week 12 endpoint for all other efficacyparameters was the Visit 7 (Week 12) measurement.

The primary efficacy analysis was performed using a 2-way analysis ofcovariance (ANCOVA) model with treatment as a factor and baseline TGvalue as a covariate. The least-squares mean, standard error, and2-tailed 95% confidence interval for each treatment group and for eachcomparison was estimated. The same 2-way ANCOVA model was used for theanalysis of secondary efficacy variables.

The primary analysis was repeated for the per-protocol population toconfirm the robustness of the results for the intent-to-treatpopulation.

The primary efficacy variable was the percent change in fasting TGlevels from baseline to Week 12. A sample size of 69 completed patientsper treatment group was expected to provide ≧90% power to detect adifference of 30% between AMR101 and placebo in percent change frombaseline in fasting TG levels, assuming a standard deviation of 45% inTG measurements and a significance level of p<0.01. To accommodate a 15%drop-out rate from randomization to completion of the double-blindtreatment period, a total of 240 randomized patients was planned (80patients per treatment group).

As shown in Table 1 and corresponding FIG. 1, subjects in the 2 g/daytreatment population exhibited a non-significant increase in total ApoC3compared to baseline (+0.3%), and a 14.3% reduction compared to placebo(p=0.0154); a non-significant increase in HDL-ApoC3 compared to baseline(+0.3%) and a 5.9% reduction compared to placebo (p=0.4062); and a 1.4%increase in non-HDL-ApoC3 compared to baseline and a 22.5% reductioncompared to placebo (p=0.0142). Subjects in the 4 g/day treatment groupexhibited reductions in total ApoC3 (10.1%), HDL-ApoC3 (21.4%) andnon-HDL-ApoC3 (5.4%) compared to baseline, which correspond tostatistically significant median placebo-adjusted reductions in totalApoC3 (25.1%, p<0.0001), HDL-ApoC3 (23.3%, p=0.002) and non-HDL-ApoC3(31.8%, p=0.0002).

Example 2 Reduction of APOC3 Levels in Subjects with Baseline FastingTriglyceride Levels of 200 mg/dl to 500 mg/dl

A multi-center, placebo-controlled, randomized, double-blind, 12-weekstudy was performed to evaluate the efficacy and safety of >96% E-EPA inpatients with fasting triglyceride levels≧200 mg/dl and <500 mg/dldespite statin therapy (the mean of two qualifying entry values neededto be ≧185 mg/dl and at least one of the values needed to be ≧200mg/dl). The primary objective of the study was to determine the efficacyof >96% E-EPA 2 g daily and 4 g daily, compared to placebo, in loweringfasting TG levels in patients with high risk for cardiovascular diseaseand with fasting TG levels≧200 mg/dl and <500 mg/dl, despite treatmentto LDL-C goal on statin therapy.

The secondary objectives of this study were the following:

-   -   1. To determine the safety and tolerability of >96% E-EPA 2 g        daily and 4 g daily;    -   2. To determine the effect of >96% E-EPA on lipid and        apolipoprotein profiles including total cholesterol (TC),        non-high-density lipoprotein cholesterol (non-HDL-C), low        density lipoprotein cholesterol (LDL-C), high density        lipoprotein cholesterol (HDL-C), and very high density        lipoprotein cholesterol (VHDL-C);    -   3. To determine the effect of >96% E-EPA on lipoprotein        associated phospholipase A₂ (Lp-PLA₂) from baseline to week 12;    -   4. To determine the effect of >96% E-EPA on low-density        lipoprotein (LDL) particle number and size;    -   5. To determine the effect of >96% E-EPA on oxidized LDL;    -   6. To determine the effect of >96% E-EPA on fasting plasma        glucose (FPG) and hemoglobin A_(1c) (HbA_(1c));    -   7. To determine the effect of >96% E-EPA on insulin resistance;    -   8. To determine the effect of >96% E-EPA on high-sensitivity        C-reactive protein (hsCRP);    -   9. To determine the effects of >96% E-EPA 2 g daily and 4 g        daily on the incorporation of fatty acids into red blood cell        membranes and into plasma phospholipids;    -   10. To explore the relationship between baseline fasting TG        levels and the reduction in fasting TG levels; and    -   11. To explore the relationship between changes of fatty acid        concentrations in plasma and red blood cell membranes, and the        reduction in fasting TG levels.

The population for this study was men and women>18 years of age with abody mass index≦45 kg/m² with fasting TG levels greater than or equal to200 mg/dl and less than 500 mg/dl and on a stable does of statin therapy(with or without ezetimibe). The statin was atorvostatin, rosuvastatinor simvastatin. The dose of statin must have been stable for ≧4 weeksprior to the LDL-C/TG baseline qualifying measurement for randomization.The statin dose was optimized such that the patients are at their LDL-Cgoal at the LDL-C/TG baseline qualifying measurements. The same statinat the same dose was continued until the study ended.

Patients taking any additional non-statin, lipid-altering medications(niacin>200 mg/day, fibrates, fish oil, other products containingomega-3 fatty acids, or other herbal products or dietary supplementswith potential lipid-altering effects), either alone or in combinationwith statin therapy (with or without ezetimibe), must have been able tosafely discontinue non-statin, lipid-altering therapy at screening.

Patients at high risk for CVD, i.e., patients with clinical coronaryheart disease (CHD) or clinical CHD risk equivalents (10-year risk>20%)as defined in the National Cholesterol Education Program (NCEP) AdultTreatment Panel III (ATP III) Guidelines were eligible to participate inthis study. Those included patients with any of the following criteria:(1) Known CVD, either clinical coronary heart disease (CHD), symptomaticcarotid artery disease (CAD), peripheral artery disease (PAD) orabdominal aortic aneurism; or (2) Diabetes Mellitus (Type 1 or 2).

Approximately 702 patients were randomized at approximately 80 centersin the U.S. The study was a 18- to 20-week, Phase 3, multi-center studyconsisting of 2 study periods: (1) A 6- to 8-week screening period thatincluded a diet and lifestyle stabilization, a non-statin lipid-alteringtreatment washout, and an LDL-C and TG qualifying period and (2) A12-week, double-blind, randomized, placebo-controlled treatment period.

During the screening period and double-blind treatment period, allvisits were within ±3 days of the scheduled time. All patients continuedto take the statin product (with or without ezetimibe) at the same dosethey were taking at screening throughout their participation in thestudy.

The 6- to 8-week screening period included a diet and lifestylestabilization, a non-statin lipid-altering treatment washout, and anLDL-C and TG qualifying period. The screening visit (Visit 1) occurredfor all patients at either 6 weeks (for patients on stable statintherapy [with or without ezetimibe] at screening) or 8 weeks (forpatients who will require washout of their current non-statinlipid-altering therapy at screening) before randomization, as follows:

-   -   Patients who did not require a washout: The screening visit        occurred at Visit 1 (Week −6). Eligible patients entered a        4-week diet and lifestyle stabilization period. At the screening        visit, all patients received counseling regarding the importance        of the National Cholesterol Education Program (NCEP) Therapeutic        Lifestyle Changes (TLC) diet and received basic instructions on        how to follow this diet.    -   Patients who required a washout: The screening visit occurred at        Visit 1 (Week −8). Eligible patients began a 6-week washout        period at the screening visit (i.e. 6 weeks washout before the        first LDL-C/TG qualifying visit). Patients received counseling        regarding the NCEP TLC diet and received basic instructions on        how to follow this diet. Site personnel contacted patients who        did not qualify for participation based on screening laboratory        test results to instruct them to resume their prior        lipid-altering medications.

At the end of the 4-week diet and lifestyle stabilization period or the6-week diet and stabilization and washout period, eligible patientsentered the 2-week LDL-C and TG qualifying period and had their fastingLDL-C and TG levels measured at Visit 2 (Week −2) and Visit 3 (Week −1).Eligible patients must have had an average fasting LDL-C level≧40 mg/dLand <100 mg/dL and an average fasting TG level≧200 mg/dL and <500 mg/dLto enter the 12-week double-blind treatment period. The LDL-C and TGlevels for qualification were based on the average (arithmetic mean) ofthe Visit 2 (Week −2) and Visit 3 (Week −1) values. If a patient'saverage LDL-C and/or TG levels from Visit 2 and Visit 3 fell outside therequired range for entry into the study, an additional fasting lipidprofile was collected 1 week later at Visit 3.1. If a third sample wascollected at Visit 3.1, entry into the study was based on the average(arithmetic mean) of the values from Visit 3 and Visit 3.1.

After confirmation of qualifying fasting LDL-C and TG values, eligiblepatients entered a 12-week, randomized, double-blind treatment period.At Visit 4 (Week 0), patients were randomly assigned to 1 of thefollowing treatment groups:

-   -   >96% E-EPA 2 g daily,    -   >96% E-EPA 4 g daily, or    -   Placebo.

226 to 234 patients per treatment group were randomized in this study.Stratification was by type of statin (atorvastatin, rosuvastatin orsimvastatin), the presence of diabetes, and gender.

During the double-blind treatment period, patients returned to the siteat Visit 5 (Week 4), Visit 6 (Week 11), and Visit 7 (Week 12) forefficacy and safety evaluations.

Eligible patients were randomly assigned at Visit 4 (Week 0) to receiveorally >96% E-EPA 2 g daily, >96% E-EPA 4 g daily, or placebo.

>96% E-EPA was provided in 1 g liquid-filled, oblong, gelatin capsules.The matching placebo capsule was filled with light liquid paraffin andcontained 0 g of >96% E-EPA. >96% E-EPA capsules were to be taken withfood (i.e. with or at the end of a meal).

During the double-blind treatment period, patients were to take 2capsules (>96% E-EPA or matching placebo) in the morning and 2 capsulesin the evening for a total of 4 capsules per day.

-   -   Patients in the >96% E-EPA 2 g/day treatment group received        1>96% E-EPA 1 g capsule and 1 matching placebo capsule in the        morning and in the evening.    -   Patients in the >96% E-EPA 4 g/day treatment group received        2>96% E-EPA 1 g capsules in the morning and evening.

Patients in the placebo group received 2 matching placebo capsules inthe morning and evening.

The primary efficacy variable for the double-blind treatment period waspercent change in TG from baseline to Week 12 endpoint. The secondaryefficacy variables for the double-blind treatment period included thefollowing:

-   -   Percent changes in total cholesterol (TC), high-density        lipoprotein cholesterol (HDL-C), LDL-C, calculated non-HDL-C,        and very low-density lipoprotein cholesterol (VLDL-C) from        baseline to Week 12 endpoint;    -   Percent change in very low-density lipoprotein TG from baseline        to Week 12;    -   Percent changes in apolipoprotein A-I (apo A-I), apolipoprotein        B (apo B), and apo A-I/apo B ratio from baseline to Week 12;    -   Percent changes in lipoprotein (a) from baseline to Week 12;    -   Percent changes in LDL particle number and size, measured by        nuclear magnetic resonance, from baseline to Week 12;    -   Percent change in remnant-like particle cholesterol from        baseline to Week 12;    -   Percent change in oxidized LDL from baseline to Week 12;    -   Changes in FPG and HbA_(1c) from baseline to Week 12;    -   Change in insulin resistance, as assessed by the homeostasis        model index insulin resistance, from baseline to Week 12;    -   Percent change in lipoprotein associated phospholipase A₂        (Lp-PLA₂) from baseline to Week 12;    -   Change in intracellular adhesion molecule-1 from baseline to        Week 12;    -   Change in interleukin-2 from baseline to Week 12;    -   Change in plasminogen activator inhibitor-1 from baseline to        Week 12. Note: this parameter will only be collected at sites        with proper storage conditions;    -   Change in hsCRP from baseline to Week 12; and    -   Change in plasma concentration and red blood cell membrane        content of fatty acid from baseline to Week 12 including EPA,        docosapentaenoic acid (DPA), docosahexaenoic acid (DHA),        arachidonic acid (AA), dihomo-γ-linolenic acid (DGLA), the ratio        of EPA/AA, ratio of oleic acid/stearic acid (OA/SA), and the        ratio of total omega-3 acids over total omega-6 acids.

Safety assessments included adverse events, clinical laboratorymeasurements (chemistry, hematology, and urinalysis), 12-leadelectrocardiograms (ECGs), vital signs, and physical examinations.

For TG, TC, HDL-C, LDL-C, calculated non-HDL-C, and VLDL-C, baseline wasdefined as the average of Visit 4 (Week 0) and the preceding lipidqualifying visit (either Visit 3 [Week −1] or if it occurs, Visit 3.1)measurements. Baseline for all other efficacy parameters was the Visit 4(Week 0) measurement.

For TG, TC, HDL-C, LDL-C, calculated non-HDL-C, and VLDL-C, Week 12endpoint was defined as the average of Visit 6 (Week 11) and Visit 7(Week 12) measurements.

Week 12 endpoint for all other efficacy parameters were the Visit 7(Week 12) measurement.

The primary efficacy analysis was performed using a 2-way analysis ofcovariance (ANCOVA) model with treatment as a factor and baseline TGvalue as a covariate. The least-squares mean, standard error, and2-tailed 95% confidence interval for each treatment group and for eachcomparison were estimated. The same 2-way ANCOVA model was used for theanalysis of secondary efficacy variables.

The primary analysis was repeated for the per-protocol population toconfirm the robustness of the results for the intent-to-treatpopulation.

Non-inferiority tests for percent change from baseline in LDL-C wereperformed between >96% E-EPA doses and placebo using a non-inferioritymargin of 6% and a significant level at 0.05.

For the following key secondary efficacy parameters, treatment groupswere compared using Dunnett's test to control the Type 1 error rate: TC,LDL-C, HDL-C, non-HDL-C, VLDL-C, Lp-PLA₂, and apo B. For the remainingsecondary efficacy parameters, Dunnett's test was be used and the ANCOVAoutput were considered descriptive.

The evaluation of safety was based primarily on the frequency of adverseevents, clinical laboratory assessments, vital signs, and 12-lead ECGs.The primary efficacy variable is the percent change in fasting TG levelsfrom baseline to Week 12. A sample size of 194 completed patients pertreatment group provided 90.6% power to detect a difference of 15%between >96% E-EPA and placebo in percent change from baseline infasting TG levels, assuming a standard deviation of 45% in TGmeasurements and a significance level of p<0.05.

Previous data on fasting LDL-C show a difference in percent change frombaseline of 2.2%, with a standard deviation of 15%, between study drugand placebo. A sample size of 194 completed patients per treatment groupprovided 80% power to demonstrate non-inferiority (p<0.05, one-sided) ofthe LDL-C response between >96% E-EPA 4 g daily and placebo, within a 6%margin. To accommodate a 10% drop-out rate from randomization tocompletion of the double-blind treatment period, a total of 648randomized patients was planned (216 patients per treatment group); 702subjects were randomized, as further described below.

Results

Of the 702 randomized subjects, 687 were in the intent-to-treat (“ITT”)population as follows:

-   -   Ultra-pure EPA, 4 g/day: 226 subjects    -   Ultra-pure EPA, 2 g/day: 234 subjects    -   Placebo: 227 subjects

Lipids were extracted from plasma and red blood cell (“RBC”) suspensionsand converted into fatty acid methyl esters for analysis using astandard validated gas chromatography/flame ionization detection method.Fatty acid parameters were compared between EPA treatment groups andplacebo using an ANCOVA model with treatment, gender, type of statintherapy, and presence of diabetes as factors, and the baseline parametervalue as a covariate. LSMs, SEs, and 2-tailed 95% confidence intervalsfor each treatment group and for each comparison were determined

Baseline characteristics of the three ITT groups were comparable, with61.4% of the ITT subjects being male, 96.3% being white, having a meanage of 61.4 years, a weight of 95.7 kg and a BMI of 32.9 kg/m². ITTsubjects with incomplete fatty acid data at baseline and/or at 12 weekswere excluded from the analyses.

As shown in Table 2 and corresponding FIG. 1, subjects in the 2 g/daytreatment population exhibited a decrease in total ApoC3 compared tobaseline (−0.6%), and an 8.5% reduction compared to placebo (p=0.0008).Subjects in the 4 g/day treatment group exhibited reductions in totalApoC3 (9.4%) compared to baseline, which corresponds to a statisticallysignificant median placebo-adjusted reduction in total ApoC3 of 19.2%(p<0.0001).

Example 3 Determination of ApoC3 in Serum or Plasma

Apolipoprotein C3 levels can be determined in serum or plasma by an invitro immunoturbidimetric immunoassay as follows.

A blood sample is centrifuged and the serum (plasma) separated. An 8 μLaliquot of the serum is combined with 300 μL of a buffer solution (e.g.,a 100 mmol/L 2-amino-2-hydroxymethyl-1,3-propanediol buffer at pH 8.5).After stabilizing at 37° C. for about 5 minutes, the difference inabsorbance at 800 nm and 340 nm is determined by a suitable analyzer(e.g., a Hitachi 7150 series Automatic Analyzer). A 100 μL aliquot of a10 U/mL anti-human apolipoprotein C3 goat-polyclonal antibody is thenadded, and the difference in absorbance at 800 nm and 340 nm isdetermined after stabilization for about 5 minutes at 37° C. Comparisonof the absorbance to a calibration curve derived from samples of knownApoC3 concentration allows calculation of the ApoC3 concentration in theblood sample.

TABLE 1 Reductions in ApoC3, HDL-ApoC3 and non-HDL-ApoC3 Placebo (N =75) AMR101 2 g/day (N = 73) AMR101 4 g/day (N=76) End-of- Change End-of-Change End-of- Change Median (%)/p-value Baseline treatment fromBaseline treatment from Baseline treatment from AMR101 AMR101 valuevalue baseline value value baseline value value baseline 4 g/day vs 2g/day vs (IQR) (IQR) % (IQR) (IQR) (IQR) % (IQR) (IQR) (IQR) % (IQR)placebo placebo Total ApoC3 26.8 32.7 12.3% 25.9 28.4 0.3% 25.6 19.7−10.1% −25.1% −14.3% (mg/dL) (17.3) (14.6) (41.5) (11.1) (12.0) (29.9)(11.6) (10.5) (27.1) <0.0001 0.0154 N = 46, 49, 53 HDL-ApoC3 14.3 15.82.9% 14.7 15.6 0.3% 12.6 10.5 −21.4% −23.3% −5.9% (mg/dL) (8.0) (7.4)(41.0) (6.1) (8.0) (29.3) (5.4) (4.1) (26.9) 0.002 0.4062 N = 39, 46, 47Non-BDL-ApoC3 11.0 12.6 22.4% 10.9 10.2 1.4% 9.3 9.3 −5.4% −31.8% −22.5%(mg/dL) (6.6) (10.2) (68.3) (5.5) (5.1) (36.9) (5.34) (4.2) (29.4)0.0002 0.0142 N = 39, 46, 47 IQR = interquartile range. ApoC3concentrations were measured using an in vitro immunoturbidimetricimmunoassay. Median values and interquartile ranges (IQR) are reportedfor all endpoint values. Baseline is defined as Visit 4 (Week 0). Theend-of-treatment value is from Visit 7 (Week 12). P-values are from theWilcoxon rank-sum test.

TABLE 2 Reductions in ApoC3, HDL-ApoC3 and non-HDL-ApoC3 Placebo (N =227) AMR101 2 g/day (N = 234) AMR101 4 g/day (N = 226) End-of- ChangeEnd-of- Change End-of- Change Median (%)/p-value Baseline treatment fromBaseline treatment from Baseline treatment from AMR101 AMR101 valueValue baseline value value baseline value value baseline 4 g/day vs 2g/day vs (IQR) (IQR) % (IQR) (IQR) (IQR) % (IQR) (IQR) (IQR) % (IQR)placebo placebo Total ApoC3 14.8 16.2 10.9% 14.8 14.8 −0.6% 15.2 13.7−9.4% −19.2% −8.5% (mg/dL) (4.5) (5.6) (30.0) (4.2) (5.0) (30.5) (4.8)(4.8) (25.9) <0.0001 0.0008 N = 201, 203, 208 IQR = interquartile range.ApoC3 concentrations were measured using an in vitro immunoturbidimetricimmunoassay. Median values and interquartile ranges (IQR) are reportedfor all endpoint values. Baseline is defined as Visit 4 (Week 0). Theend-of-treatment value is from Visit 7 (Week 12). P-values are from theWilcoxon rank-sum test.

What is claimed is:
 1. A method of reducing an apolipoprotein C-IIIlevel of a subject having fasting baseline triglycerides of at leastabout 500 mg/dl, the method comprising administering to the subject apharmaceutical composition comprising about 1 g to about 4 g of ethyleicosapentaenoate per day, wherein the apolipoprotein C-III level isreduced by at least about 5%.
 2. The method of claim 1, wherein theapolipoprotein C-III level is reduced by at least about 25%.
 3. Themethod of claim 1, wherein about 2 g per day of ethyl eicosapentaenoateis administered to the subject.
 4. The method of claim 1, wherein about3.8 g to about 4 g per day of ethyl eicosapentaenoate is administered tothe subject.
 5. The method of claim 1, wherein the pharmaceuticalcomposition is administered to the subject daily for a period of 1 toabout 12 weeks.
 6. The method of claim 1, wherein the reduction in APOC3level is in comparison to control subjects having fasting triglyceridesof at least about 500 mg/dl who have not received the pharmaceuticalcomposition.
 7. The method of claim 1, wherein the pharmaceuticalcomposition comprises at least about 95%, by weight of all fatty acidspresent, ethyl eicosapentaenoate.
 8. The method of claim 1, wherein thepharmaceutical composition comprises no more than about 10%, by weightof all fatty acids present, docosahexaenoic acid or a derivativethereof, if any.
 9. The method of claim 1, wherein the pharmaceuticalcomposition comprises at least about 95%, by weight of all fatty acidspresent, ethyl eicosapentaenoate and no more than about 2%, by weight ofall fatty acids present, docosahexaenoic acid or a derivative thereof,if any.